Various methods and devices exist for separating component parts of a sample fluid to obtain target particles. These methods include filters, centrifuges, chromatographs, and other well-known fluid separation methods. Other apparatus and methods exist for separating a particular cell subpopulation, or target cells, from a mixture of cells. These methods include gross separation using columns, centrifuges, filters, separation by killing of unwanted cells, separation with fluorescence activated cell sorters, separation by directly or indirectly binding cells to a ligand immobilized on a physical support, such as panning techniques, separation by column immunoadsorption, and separation using magnetic immunobeads. Such immunoselection methods have been used to either positively or negatively select target cells, wherein positive selection refers to the direct selection and recovery of specific target cells, while negative selection refers to the elimination of a specific target cell subpopulation from a heterogeneous population of cells.
Columns are common to the above described methods for general separation of target particles from a sample fluid and for the more specific immunoselection methods for selecting target cells from a sample fluid. Typically, a column has an entrance end, an exit end, and a substrate positioned intermediate the entrance and exit ends. In operation, the sample fluid is provided to the entrance end of the column and is moved through the column under pressure. As the fluid passes through the column, the substrate separates the target particle from the fluid composition so that the target particle exiting the column will be substantially pure. The target particle exiting the column is collected and retained as the collected product of the separation. Accordingly, the substrate is selected for a particular separation to separate the target particle from the sample fluid.
Various substrates exist for use with columns to separate the target particle from the sample fluid. Generally, the type of substrate selected for performing the separation will determine how the target particles are separated from the sample fluid. As an example, with substrates typically used in chromatography, the sample fluid is forced through the column under pressure using a solvent solution. The substrate is selected so that the target particles exhibit substantially different binding characteristics with the substrate than the remaining components of the sample fluid so that the time necessary for the target particles to pass through the substrate will be substantially different from the time necessary for the remaining components of the sample fluid to pass through the substrate. Accordingly, a substantially pure composition of the target particles will exit the column at a predetermined time for collection.
Referring again to immunoselection methods using column immunoadsorption devices, the substrate may contain beads that have been coated with a ligand, such as an antibody, immobilized on the surface of the beads. The ligands are selected to bind with the target particles, thereby immobilizing the target particles within the column. After an adequate amount of the fluid composition has been passed through the column to saturate the beads of the column substrate, the target particles can be liberated from the beads using various techniques. Preferably, the target particles are liberated from the beads by gently agitating the beads to break the bond between the target particles and the immobilized ligand. However, existing columns for performing immunoadsorption have proven undesirable since they have failed to provide commercially acceptable apparatus for agitating the substrate to aid in liberating the target particles. The prior art devices have also failed to provide apparatus for controlling the amount of agitation provided to the substrate, to prevent damage to the target particles and are further undesirable for this reason.
Other column separation devices, and particularly other column immunoadsorption devices, have also proven inefficient since these devices require considerable intervention from the operator to control the introduction of the sample fluid to the column as well as controlling the withdrawal of the target particle from the column. Typically, column separation devices must be actively monitored through various stages including stages for cleansing the column prior to introduction of the sample fluid and stages for passing the sample fluid through the column. These stages generally require significant intervention from the operator to perform each of the foregoing steps of the fluid separation process, and to perform substeps within these steps. Accordingly, the efficiency of these devices is necessarily limited by the skill and effectiveness of the operator controlling the process. It is desirable, therefore, to provide apparatus for performing fluid separation that minimizes the amount of intervention necessary from an operator of the apparatus. Additionally, it is desirable to provide apparatus for performing fluid separation wherein the movement of fluid through the apparatus may be precisely controlled by the separation apparatus without significant intervention by the operator.